X-ray Free Electron Lasers
With peak intensities that may be 108-1010 times greater than those of synchrotron sources, the X-ray free electron laser (“XFEL”) has enabled novel methodological advances in biological imaging via serial femtosecond crystallography (“SFX”). In contrast to conventional X-ray protein crystallography where samples are cryogenically prepared to mitigate radiation damage, SFX probes protein crystals that are suspended in their mother liquor. They may be continuously injected across a pulsed X-ray beam. X-ray pulses are so brief that radiation damage may be outrun. Information about the unperturbed structure in its native environment is captured before X-ray damage has any effect on the sample, such that acquired structure data more reliably reflects the biologically active state of the protein sample. Furthermore, protein crystals that are too small and/or sensitive to radiate, including delicate human membrane proteins, may be imaged using SFX.
Gas Dynamic Virtual Nozzles
Continuously transporting millions of environmentally sensitive protein crystals through high vacuum and into the pathway of the X-ray beam may be accomplished with the Gas Dynamic Virtual Nozzle (“GDVN”), which may be capable of delivering a continuous, steady supply of hydrated sample in UHV for several hours without clogging.
For example, if the tapered end of a straight-bore capillary is placed in a focused gas sheath the emerging liquid forms may form a jet that subsequently breaks up into droplets due to Rayleigh-Plateau instability. Unlike conventional solid-walled nozzles, the walls of this “virtual” nozzle are made of gas, thereby reducing clogging.
GDVN Fabrication
To fabricate such an injector, the end of a small length of glass capillary may be flame polished and grounded to create a converging inner profile and beveled exterior. The opposite end of this capillary may be inserted and glued into a section of stainless steel tubing, so that the modified end of the glass capillary protrudes several millimeters. A smaller, polyimide-coated, glass capillary 140ith its end grounded into a truncated cone may be inserted into this apparatus and brought into close proximity with the converging section at the modified end of the outer glass capillary. Axial centering of the capillary may be accomplished with a spacer as shown in FIG. 6b or by using an outer capillary 140ith square cross section.
GDVN's typically operate with pressurized gas, where one gas source drives the sample of interest down the inner capillary and the other gas source flows coaxially through the outer glass capillary and may be geometrically focused near the tip. The sample emerges from the conical tip of inner capillary directly into this gas-focusing region and may thereby be reduced in size by a factor of 10 or more; typically a 50 micron emergent stream is reduced gas-dynamically to a jet of about 1 micron to about 5 microns. This jet may leave the gas aperture as a freely suspended stream, remaining liquefied even in a high vacuum environment (FIG. 6a).
Submicron sized jets, which are of importance to the development of single particle imaging methods, have been realized by adjusting the capillary position and gas aperture geometry so that the cone of the inner capillary tip is flush with or protrudes slightly outside of the gas aperture, placing the emerging stream directly into the supersonic flow of the free-jet expansion produced while exhausting into vacuum.
Despite having been used effectively to date in a number of protein imaging experiments, improvements to the fabrication process may be desired. The artisan-like fabrication method described above typically results in irreproducible nozzles. Each nozzle is unique in its reliability, capability, and operating parameters. And not every nozzle therefore may perform as desired.
GDVN jetting may be sensitive to slight asymmetry in the modified end of the outer capillary and/or misalignment of the central capillary line thereto, leading to undesirable behavior such as off-axis jetting and instability. This may be problematic during SFX experiments. Unstable jets may be difficult to hit or target with the X-ray, reducing data collection rates. Off-axis jetting in vacuum environments typically leads to ice formation as the jet hitting a nearby wall may freeze and grow back along the stream until the nozzle is rendered inoperable. Furthermore, ice formation near the nozzle tip may present a problem for sensitive detection equipment. For example, if the intensity of the X-ray beam is maximized for a weakly diffracting sample, the sudden introduction of strongly diffracting ice can cause damage to the detector. Consequently, nozzle fabrication typically requires great skill and time to produce reliable jets for an increasingly large and complex experimental demand.
Microfabrication Techniques
Improved fabrication may be needed to achieve reliable jets consistently. Many microfabrication techniques exist that offer greater resolution, repeatability, and yield. Soft photolithography has recently been used to fabricate functional polydimethylsiloxane (PDMS) GDVN devices. Injection molding has been used to replace the modified end of the outer glass capillary 140ith a strong, high-resolution ceramic material. However, PDMS GDVN devices, while simple and functional, may not be sufficiently durable under extreme pressure conditions, and injection-molded gas apertures may still require manual positioning of the inner glass capillary. A technique is desired to build all GDVN components in robust material using a single process. Direct laser writing may offer the ability to write both the internal and external GDVN structures at high resolution in robust materials with write times on the order of hours.
3D Printing Gas Dynamic Virtual Nozzles
2-Photon Polymerization (2PP) is a direct-write non-linear absorption process that may be capable of patterning structures with resolution well beyond the diffraction limit. By tightly focusing a laser in a medium that is sensitive to only higher order effects, just a small region near the beam focus may be polymerized. Features as small as 100 nm to 150 nm may be made reproducibly. By moving the absorbing medium relative to the laser in a controlled manner, as in with a computer generated design, complex high resolution structures may be realized. In principle this allows all GDVN components to be formed using a single process at high resolution.
However, challenges in making 2PP a viable printing technology for the GDVN may arise due to the extreme differences in scale. For example, the relatively large size of a traditional GDVN may make submicron resolution scanning a prohibitively long process in the absence of specialty fast-writing stages. The Photonic Professional GT from Nanoscribe GmbH may be used to meet these device-specific challenges. It utilizes a 3-dimensional piezoelectric stage and galvo mirror system to achieve fast write times (e.g., 10 mm/s and 100 μm/s respectively) over a volume of approximately 300×300×300 cubic microns. These fast-write volumetric units may be stitched together with standard motorized stages. Working in concert for the particular application, these systems may allow writing times that are much faster than the norm.
Fabrication
Nozzle Design
The nozzle may be designed using standard CAD software. In order to minimize the write time the 3D printed GDVN may be designed to be as small as possible, taking into consideration the need for the device to withstand both high and low pressure extremes as well as the need for feasibility in the manner of coupling the device to supply lines. Furthermore, the various 3D printed channels may be made to be as open as possible while maintaining structural integrity. This may be desirable to aid in the successful removal of uncured photoresist from very small closed channels.
3D Printing
The printing system was successfully adapted to use a larger objective focusing lens and a corresponding resist formula. This resulted in lower resolution (500 nm) but faster writing, since a larger volume was traced out in the same amount of time. The system produced 400×400×400 cubic micron fast-write units. This allowed the nozzles described herein to be written in under 4 hours. As a comparison, an earlier iteration of similar size took 24 hours to write using the higher resolution objective lens.
The stitched interfaces between the 400×400×400 μm3 fast-written units were visible with optical microscopy as vertical parabolic cross-sections through the cone of the nozzle. Additional stitching interfaces, presumably between vertical motor advancements, were seen as thin horizontal slices spaced about 10 μm apart (FIG. 7a). Optical microscopy of nozzles immersed in a nearly index-matched medium (Glycerol) gave an undistorted view of both types of stitching as projected through the nozzle (FIG. 7b). The absolute positioning error for the horizontal slices was almost undetectable with optical microscopy, suggesting submicron error. However, disruptions in the continuity of sidewall profiles were visible as 1 to 2 micron-sized dips at the edge of each horizontal stitched region (FIG. 7b inset). Positioning errors between 400×400×400 μm3 fast-written units were more apparent, ranging from 1 to 5 μm (FIG. 7a, 7b inset).
Connecting Sample Lines
The sample lines were glued into place using a custom mounting stage with a commercial micromanipulator. Gluing of the inner capillary 140 as achieved by first inserting the capillary into the receiving port of the printed nozzle. A small drop of fast-curing epoxy was placed on the end of a hypodermic needle that was attached to the micromanipulator. The tip of the needle was brought near to the gluing target with preprogrammed coordinates and then controlled manually for the gluing process. The “glue guide” functioned according to its design to draw a single drop of applied epoxy completely around the inserted capillary using capillary forces (FIG. 8a). The epoxy bond between the inner capillary and the printed nozzle was allowed to fully cure overnight before adhering it to the outer gas transport tube. The end of the gas transport tube was grinded to match the angular relief of the printed nozzle. Careful design of the nozzle dimensions allowed the nozzle to be inserted into the gas transport tube and held in place by friction. Epoxy was then applied to the interface using the micromanipulator, and distributed by rotating the gas transport tube relative to the fixed micromanipulator applicator position (FIG. 8b). For testing purposes a thin-walled glass capillary 140 as placed between the nozzle and a different stainless steel gas transport tube of slightly larger inner diameter, similar to what is done with conventional GDVN fabrication. However, in practice, a direct connection from the printed nozzle to the stainless steel gas transport tube is preferred for rigidity. Helium gas was supplied to the gas transport tube through a second glass capillary that was inserted into the gas transport tube. Epoxy was applied by hand to the rear end of the tube in order to provide a gastight seal and to fix all capillaries in place with respect to each other. After the epoxy was fully cured the stainless steel tube was attached to the standard GDVN holder apparatus, which in turn has connections to the nozzle rods used for in-vacuum sample injection.
Results
In-Lab Testing
Three nozzles were tested with pure water and helium gas to determine whether the printing resolution and symmetry were sufficient to produce a straight jet and to test whether the nozzles performed in a reproducible manner without fracturing. Initial observations revealed that the nozzles did not jet straight in either vacuum or atmosphere, that the nozzles required very little helium gas pressure to operate compared to the traditional GDVN with a 2 meter long, 100 μm ID gas supply capillary at a flow rate of 5 μl/min (<50 psi compared to about 300 psi as measured upstream of the supply capillary), and that the angular deviation of the jet trajectory from the nozzle axis was highly dependent on helium gas pressure. Comparative tests of the three nozzles were performed by taking videos of each nozzle jetting while being rotated slowly about the nozzle axis. For these tests helium gas pressure, water flow rate, and supply capillary length were made identical for the testing of each nozzle by never adjusting the gas regulator position between nozzle changes, using an in-line digital liquid flow meter, and cutting the capillaries to equal lengths within 1 cm. Imaging points during rotation were chosen at 60-degree intervals based on the centering of the cylindrical outer sidewall of the nozzle as shown in FIG. 9. Collections of these images in sequence were compared side by side and matched according to the angular deviation of the jet from the nozzle axis (FIG. 9). The result was that the nozzles did jet reproducibly, as shown in FIG. 9.
Serial Crystallography
Two nozzles were used in actual serial crystallography experiments (SFX) at the CXI station at the Linac Coherent Light Source (LCLS, a free-electron X-ray laser) to test whether or not the resist material showed any adverse effects. Background produced by the nozzles when in close proximity with the x-ray beam was measured at 10% and 100% transmission. Crystal screening was performed using the nozzles for two membrane proteins. A snapshot diffraction pattern obtained with a printed nozzle at CXI is shown in FIG. 10, with sharp Bragg spots extending to 4 Å resolution. This pattern was collected with 8.7 keV, using 40 fs XFEL pulses, with a distance of 138 mm between sample and detector. No evidence was observed that the printed nozzles affected diffraction quality compared to patterns obtained from the same samples using standard GDVN nozzles. Overall, the jets appeared to perform very similarly in comparison with the standard glass capillary-tube GDVN.
Discussion and Conclusion
The images collected in FIG. 9 suggest that the nozzles are reproducible to a very reasonable approximation. It is important to consider that small variations in this reproducibility may be attributable to factors other than the quality of the printed nozzles themselves. These factors include reproducibility of the gluing process, the apparatus upstream of the nozzle, and the operating parameters for each nozzle. Experimental errors also play a role, such as the perpendicularity of the microscope camera to the nozzle, which tends to vary as the nozzle is rotated. Another consideration is that the sequence of six images for each nozzle taken during rotation was performed without a universal starting point since the appearances of the nozzle at 120-degree rotations are essentially indistinguishable. Although comparing the sequences side by side quickly lead to the matching of the sequences, printing distinctive markers at 60-degree intervals on the nozzles themselves would have more conclusively showed reproducibility.
The fact that the nozzles jet off-axis in a reproducible way suggests that there is a systematic flaw in the printing process, such as a slight skew along the nozzle's nominal symmetry axis. In this case the nozzles themselves could in principle be used to calibrate the printing machine, or the computer-generated design itself could be modified to correct for the error in the printing process. Another possible reason for off-axis jetting is that the nozzle design itself is not optimized for stable jetting, a condition that would need to be verified with additional studies. The potentially gas permeable stitching interfaces may also play a role in redirecting the jet. Another possibility is the hardening of residual photoresist left behind due to incomplete chemical development, which was observed just upstream of the tip of the inner nozzle structure in the complete version of the image shown in FIG. 7b (essentially not pictured in FIG. 7b). However, if these depositions cause off-axis jetting, we would expect that the essentially random deposits of extra material would lead to irreproducible jets.
In actual experimentation the off-axis jetting did not appear to impair data collection. It was confirmed that the photoresist material itself does not appear to adversely affect the protein crystals. In most respects, the printed nozzle performance was no different than the performance of standard GDVN's, apart from the noticeable improvement in reproducibility when compared to hand-crafted nozzles.
This successful acquisition of diffraction data in serial crystallography experiments indicates that 2PP 3D printed nozzles can be used in place of glass capillary GDVN systems with the immediate advantage of reproducible nozzle fabrication for rapid prototyping and development of new types of jets. Further work is expected to provide optimized dimensions for best performance, and a range of other jets for other purposes, such as solution scattering and possibly single-particle imaging.